US12571059B2 - Compositions and methods for detection of Epstein Barr virus (EBV) - Google Patents

Compositions and methods for detection of Epstein Barr virus (EBV)

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US12571059B2
US12571059B2 US17/597,725 US202017597725A US12571059B2 US 12571059 B2 US12571059 B2 US 12571059B2 US 202017597725 A US202017597725 A US 202017597725A US 12571059 B2 US12571059 B2 US 12571059B2
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ebv
nucleic acid
primers
seq
sample
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Aaron T. Hamilton
Marintha Heil
Debra Liggett
Jingtao Sun
Ling Wang
Xiaoning Wu
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Roche Sequencing Solutions Inc
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Roche Sequencing Solutions Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • EBV viral load measurement is becoming a routine test for managing transplant recipients as well as diagnosing and following EBV-associated diseases.
  • serial DNA measurements can be used to indicate the need for potential treatment changes and to assess viral response to treatment.
  • EBV is a DNA virus with multiple well-conserved target regions for assay design, the possibility of un-encountered substitutions or deletions meant that a dual-target approach was prudent and sensible.
  • the design of the primers and probes used available public sequence information to maximize inclusivity for EBV and exclude other herpesviruses.
  • This invention consists of novel oligonucleotide designs, designed to maximize inclusivity of EBV and prevent cross reactivity with other templates.
  • This EBV assay may be used on the cobas® 6800/8800.
  • the primers and probes of the present invention may be used as a dual target assay, with both targets using the same dye, to safeguard against viral sequence heterogeneity.
  • the design strategy was to select conserved sequence regions from the EBV genome and assess several primer and probe combinations for each target.
  • the five regions from which assay designs were selected were the IR-1 repeat, LMP2A, EBNA-1 (BKRF-1), BMRF-2, and Reductase/BORF-2.
  • Sequences included both type 1 and type 2 EBV, and sequences from worldwide geographic locations including Asia, Africa, North America, South America, Europe and Australia. After a screening and optimization process, the two candidates were from just beyond the final exon of EBNA-1 and from within the coding sequence of BMRF-2, were identified. These candidates could be used individually or together duplexed in a dual target assay.
  • two sets of primers and probe are employed (each set detecting either EBNA-1 or BMRF-2).
  • Certain embodiments in the present disclosure relate to methods for the rapid detection of the presence or absence of EBV in a biological or non-biological sample, for example, multiplex detection and quantitating of EBV by real-time polymerase chain reaction (PCR) in a single test tube or vessel.
  • Embodiments include methods of detection of EBV comprising performing at least one cycling step, which may include an amplifying step and a hybridizing step.
  • embodiments include primers, probes, and kits that are designed for the detection of EBV in a single tube or vessel.
  • One aspect of the invention is directed to a method for detecting one or more target nucleic acids of Epstein Barr Virus (EBV) in a sample, the method comprising: (a) providing a sample; (b) performing an amplification step comprising contacting the sample with one or more set of primers to produce an amplification product, if the one or more target nucleic acids of EBV is present in the sample; (c) performing a hybridization step, comprising contacting the amplification product, if the one or more target nucleic acids of EBV is present in the sample, with one or more probes; and (d) performing a detection step, comprising detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of the one or more target nucleic acids of EBV in the sample, and wherein the absence of the amplification product is indicative of the absence of the one or more target nucleic acids of EBV in the sample; and wherein the one or more set of primers and the one or more
  • the sample is a biological sample.
  • the biological sample is plasma.
  • the biological sample is blood.
  • the method is for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, wherein the one or more set of primers and the one or more probes for detecting the first target nucleic acids of EBV comprises: (i) a set of primers comprising a nucleic acid sequence of SEQ ID NOs:1 and 3, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and wherein the one or more set of primers and the one or more probes for detecting the second target nucleic acids of EBV comprises: (ii) a set of primers comprising a nucleic acid sequence of SEQ ID NO:4 and 6, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:3, or
  • first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are not overlapping.
  • Another aspect is directed to a method for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, the method comprising: (a) providing a sample; (b) performing an amplification step comprising contacting the sample with two sets of primers to produce an amplification product, if the one or more target nucleic acids of EBV is present in the sample; (c) performing a hybridization step, comprising contacting the amplification product, if the one or more target nucleic acids of EBV is present in the sample, with two probes; and (d) performing a detection step, comprising detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of the one or more target nucleic acids of
  • the first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are not overlapping.
  • the sample is a biological sample. In another embodiment, the biological sample is plasma. In another embodiment, the biological sample is blood.
  • kits for detecting one or more target nucleic acids of EBV comprising amplification reagents comprising: (a) a DNA polymerase; (b) nucleotide monomers; (c) one or more set of primers; and (d) one or more probes, wherein the one or more set of primers and the one or more probes comprise: (i) a set of primers comprising a nucleic acid sequence of SEQ ID NOs:1 and 3, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and/or (ii) a set of primers comprising a nucleic acid sequence of SEQ ID NO:4 and 6, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:3, or a complement thereof.
  • the one or more probes is labeled with a donor fluorescent moiety and a corresponding acceptor moiety.
  • the kit is for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, wherein the one or more set of primers and the one or more probes for detecting the first target nucleic acids of EBV comprises: (i) a set of primers comprising a nucleic acid sequence of SEQ ID NOs:1 and 3, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and wherein the one or more set of primers and the one or more probes for detecting the second target nucleic acids of EBV comprises: (ii) a set of primers comprising a nucleic acid sequence of SEQ ID NO:4 and 6, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:3, or a complement thereof.
  • kits for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample comprising amplification reagents comprising: (a) a DNA polymerase; (b) nucleotide monomers; (c) a first set of primers and one probe for detecting a first target nucleic acid of EBV; and (d) a second set of primers and one probe for detecting a second target nucleic acid of EBV, wherein one first set of primers and one probe for detecting a first target nucleic acids of EBV comprises: (i) a set of primers comprising a nucleic acid sequence of SEQ ID NOs:1 and 3, or complements thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and wherein the second set of primers and one probe for detecting a second target nucleic acids of EBV comprises: (ii) a set of primers comprising a nucleic acid sequence
  • the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.
  • the first target nucleic acid of EBV and the second target nucleic acid of EBV are different.
  • the first target nucleic acid of EBV and the second target nucleic acid of EBV are not overlapping.
  • an oligonucleotide comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs:1-6, or a complement thereof, which oligonucleotide has 100 or fewer nucleotides.
  • the present disclosure provides an oligonucleotide that includes a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:1-6 , or a complement thereof, which oligonucleotide has 100 or fewer nucleotides.
  • these oligonucleotides may be primer nucleic acids, probe nucleic acids, or the like in these embodiments.
  • the oligonucleotides have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.)
  • the oligonucleotides comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability relative to unmodified nucleotides.
  • the oligonucleotides comprise at least one label and optionally at least one quencher moiety.
  • the oligonucleotides include at least one conservatively modified variation.
  • “Conservatively modified variations” or, simply, “conservative variations” of a particular nucleic acid sequence refers to those nucleic acids, which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • One of skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
  • amplification can employ a polymerase enzyme having 5′ to 3′ nuclease activity.
  • the donor fluorescent moiety and the acceptor moiety e.g., a quencher
  • the acceptor moiety may be within no more than 5 to 20 nucleotides (e.g., within 7 or 10 nucleotides) of each other along the length of the probe.
  • the probe includes a nucleic acid sequence that permits secondary structure formation. Such secondary structure formation may result in spatial proximity between the first and second fluorescent moiety.
  • the second fluorescent moiety on the probe can be a quencher.
  • the present disclosure also provides for methods of detecting the presence or absence of EBV or EBV nucleic acid, in a biological sample from an individual. These methods can be employed to detect the presence or absence of EBV nucleic acid in plasma, for example, for use in blood screening and diagnostic testing. Additionally, the same test may be used by someone experienced in the art to assess urine and other sample types to detect and/or quantitate EBV nucleic acid. Such methods generally include performing at least one cycling step, which includes an amplifying step and a dye-binding step.
  • the amplifying step includes contacting the sample with a plurality of pairs of oligonucleotide primers to produce one or more amplification products if a nucleic acid molecule is present in the sample, and the dye-binding step includes contacting the amplification product with a double-stranded DNA binding dye.
  • Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye into the amplification product, wherein the presence of binding is indicative of the presence of EBV nucleic acid in the sample, and wherein the absence of binding is indicative of the absence of EBV nucleic acid in the sample.
  • a representative double-stranded DNA binding dye is ethidium bromide.
  • nucleic acid-binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green.
  • methods also can include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, wherein the melting temperature confirms the presence or absence of EBV nucleic acid.
  • kits for detecting and/or quantitating one or more nucleic acids of EBV can include one or more sets of primers specific for amplification of the gene target; and one or more detectable oligonucleotide probes specific for detection of the amplification products.
  • the kit can include probes already labeled with donor and corresponding acceptor moieties, e.g., another fluorescent moiety or a dark quencher, or can include fluorophoric moieties for labeling the probes.
  • the kit can also include nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase.
  • the kit can also include a package insert and instructions for using the primers, probes, and fluorophoric moieties to detect the presence or absence of EBV nucleic acid in a sample.
  • FIG. 3 shows the EBV targets and oligonucleotide set for BMRF2, including the 98 base pair amplicon generated by the primers, and detected by the probe.
  • FIG. 3 depicts that the forward primer has a sequence of SEQ ID NO: 1, the reverse primer has a sequence of SEQ ID NO:3, and the probe has a sequence of SEQ ID NO:2.
  • FIGS. 4 A-C shows PCR growth curves of a dilution series of the performance of the assay on a Qnostics EBV Analytic Panel (Qnostics Catalog No. EBVAQP03-C) versus a WHO Standard for EBV (NIB SC code 09/260), at 8 ( FIG. 4 C ), 80 ( FIG. 4 B ), and 8,000 IU/ml ( FIG. 4 A ).
  • Other EBV sample types were also tested, such as extracted EBV DNA and Raji cell line DNA spiked into EBV-negative plasma (data not shown).
  • FIGS. 5 A- 5 L shows PCR growth curves of a dilution series of the performance of the multiplexed assay on a cell culture derived material (from 1E4 IU/mL to 20 IU/mL) ( FIGS. 5 H- 5 L ) and for a control template (from 1E9 IU/mL to 1E3 IU/mL) ( FIGS. 5 A- 5 G ).
  • Channel 2 shows the dual target EBV assay and channel 5 is an internal control reaction.
  • FIGS. 6 A and 6 B show PCR growth curves for separate reactions for EBNA1 ( FIG. 6 A and BMRF2 ( FIG. 6 B on plasmid templates containing individual targets (E1c, B2c) or both targets (dC), as well as a B95-8 cell line extraction (containing copies of EBV DNA). These studies demonstrate that the dual target EBNA1 and BMRF2 EBV assays are efficient and specifically identifying EBNA1 and BMRF2 EBV nucleic acid.
  • FIG. 7 shows Linearity data for the dual target assay on a genotype 1 cell culture derived material and for a control template.
  • FIG. 8 shows Linearity data for the dual target assay on a genotype 2 EBV-containing cell culture derived material.
  • Diagnosis of EBV infection by nucleic acid amplification provides a method for rapidly, accurately, reliably, specifically, and sensitively detecting and/or quantitating the EBV infection.
  • a real-time PCR assay for detecting and/or quantitating EBV nucleic acids, including DNA and/or RNA, in a non-biological or biological sample is described herein.
  • Primers and probes for detecting and/or quantitating EBV are provided, as are articles of manufacture or kits containing such primers and probes.
  • the present disclosure includes oligonucleotide primers and fluorescent labeled hydrolysis probes that hybridize to the EBV genome, in order to specifically identify EBV using, e.g., TaqMan® amplification and detection technology.
  • the disclosed methods may include performing at least one cycling step that includes amplifying one or more portions of the nucleic acid molecule gene target from a sample using one or more pairs of primers.
  • “EBV primer(s)” as used herein refer to oligonucleotide primers that specifically anneal to nucleic acid sequences found in the EBV genome, and initiate DNA synthesis therefrom under appropriate conditions producing the respective amplification products.
  • Examples of nucleic acid sequences found in the EBV genome include nucleic acids within viral capsid protein region of the EBV genome, such as the VP2 region.
  • Each of the discussed EBV primers anneals to a target such that at least a portion of each amplification product contains nucleic acid sequence corresponding to the target.
  • the one or more amplification products are produced provided that one or more nucleic acid is present in the sample, thus the presence of the one or more amplification products is indicative of the presence of EBV in the sample.
  • the amplification product should contain the nucleic acid sequences that are complementary to one or more detectable probes for EBV.
  • “EBV probe(s)” as used herein refer to oligonucleotide probes that specifically anneal to nucleic acid sequences found in the EBV genome.
  • Each cycling step includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with the one or more detectable EBV probes for detection of the presence or absence of EBV in the sample.
  • amplifying refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid molecule (e.g., nucleic acid molecules from the EBV genome).
  • Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product.
  • Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g., MgCl 2 and/or KCl).
  • a DNA polymerase enzyme e.g., Platinum® Taq
  • an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme e.g., MgCl 2 and/or KCl.
  • primer refers to oligomeric compounds, primarily to oligonucleotides but also to modified oligonucleotides that are able to “prime” DNA synthesis by a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g., oligonucleotide provides a free 3′-OH group where further “nucleotides” may be attached by a template-dependent DNA polymerase establishing 3′ to 5′ phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.
  • hybridizing refers to the annealing of one or more probes to an amplification product.
  • Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.
  • 5′ to 3′ nuclease activity refers to an activity of a nucleic acid polymerase, typically associated with the nucleic acid strand synthesis, whereby nucleotides are removed from the 5′ end of nucleic acid strand.
  • thermostable polymerase refers to a polymerase enzyme that is heat stable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3′ end of each primer and proceeds in the 5′ to 3′ direction along the template strand.
  • Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished, if necessary.
  • nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.
  • nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase that typically adds nucleotides at the 3′ terminal end of a nucleic acid.
  • a nucleotide incorporating biocatalyst such as a polymerase that typically adds nucleotides at the 3′ terminal end of a nucleic acid.
  • nucleic acid sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection.
  • sequence comparison algorithms available to persons of skill or by visual inspection.
  • Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet.
  • modified nucleotide in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide.
  • Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, e.g., a t-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a 7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU
  • modified nucleotide substitutions modify melting temperatures (Tm) of the oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides.
  • Tm melting temperatures
  • certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and/or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Pat. No. 6,001,611, which is incorporated herein by reference.
  • Other modified nucleotide substitutions may alter the stability of the oligonucleotide, or provide other desirable features.
  • the present disclosure provides methods to detect EBV by amplifying, for example, a portion of the EBV nucleic acid sequence. Specifically, primers and probes to amplify and detect and/or quantitate EBV nucleic acid molecule targets are provided by the embodiments in the present disclosure.
  • Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means.
  • One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured).
  • the heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° C. to about 105° C. for a time depending on features of the reaction such as temperature and the nucleic acid length.
  • Denaturation is typically performed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5 min).
  • FRET technology is based on a concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and/or quantitated.
  • the donor typically transfers the energy to the acceptor when the donor is excited by light radiation with a suitable wavelength.
  • the acceptor typically re-emits the transferred energy in the form of light radiation with a different wavelength.
  • non-fluorescent energy can be transferred between donor and acceptor moieties, by way of biomolecules that include substantially non-fluorescent donor moieties (see, for example, U.S. Pat. No. 7,741,467).
  • corresponding refers to an acceptor fluorescent moiety or a dark quencher having an absorbance spectrum that overlaps the emission spectrum of the donor fluorescent moiety.
  • the wavelength maximum of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.
  • Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency Foerster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength.
  • a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety.
  • a corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum (>600 nm).
  • Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid, 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.
  • acceptor fluorescent moieties depending upon the donor fluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of Lanthanide ions (e.g., Europium, or Terbium).
  • Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).
  • the donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm.
  • the length of each linker arm is important, as the linker arms will affect the distance between the donor and acceptor fluorescent moieties.
  • the length of a linker arm can be the distance in Angstroms ( ⁇ ) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 ⁇ to about 25 ⁇ .
  • the linker arm may be of the kind described in WO 84/03285.
  • WO 84/03285 also discloses methods for attaching linker arms to a particular nucleotide base, and also for attaching fluorescent moieties to a linker arm.
  • the present disclosure provides methods for detecting the presence or absence of EBV in a biological or non-biological sample.
  • Methods provided avoid problems of sample contamination, false negatives, and false positives.
  • the methods include performing at least one cycling step that includes amplifying a portion of EBV target nucleic acid molecules from a sample using one or more pairs of EBV primers, and a FRET detecting step. Multiple cycling steps are performed, preferably in a thermocycler. Methods can be performed using the EBV primers and probes to detect the presence of EBV, and the detection of EBV indicates the presence of EBV in the sample. As described herein, amplification products can be detected using labeled hybridization probes that take advantage of FRET technology.
  • One FRET format utilizes TaqMan® technology to detect the presence or absence of an amplification product, and hence, the presence or absence of EBV.
  • TaqMan® technology utilizes one single-stranded hybridization probe labeled with, e.g., one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent.
  • one fluorescent moiety or dye e.g., HEX or FAM
  • quencher e.g., BHQ-2
  • the second moiety is generally a quencher molecule.
  • the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded by the 5′ to 3′ nuclease activity of, e.g., the Taq Polymerase during the subsequent elongation phase.
  • the fluorescent moiety and the quencher moiety become spatially separated from one another.
  • the fluorescence emission from the first fluorescent moiety can be detected.
  • the presence of FRET indicates the presence of EBV in the sample
  • the absence of FRET indicates the absence of EBV in the sample.
  • Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of a test result, however.
  • Representative biological samples that can be used in practicing the methods include, but are not limited to whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissue infections. Collection and storage methods of biological samples are known to those of skill in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and/or kits known in the art) to release EBV nucleic acid or in some cases, the biological sample can be contacted directly with the PCR reaction components and the appropriate oligonucleotides. In some instances, the biological sample is whole blood.
  • nucleic acids within the whole blood undergo considerable amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that will lyse, denature, and stabilize whole blood components, including nucleic acids, such as a nucleic acid-stabilizing solution. In such cases, the nucleic acids can be better preserved and stabilized for subsequent isolation and analysis, such as by nucleic acid test, such as PCR.
  • nucleic acid-stabilizing solution are well known in the art, including, but not limited to, cobas PCR media, which contains 4.2 M guanadinium salt (GuHC1) and 50 mM Tris, at a pH of 7.5.
  • the primers and probes for the EBV test were designed by seeding primers and probes along the genome in the most conserved regions based on the alignment.
  • One set of oligonucleotides (SEQ ID NOs:1-3) was designed to detect the BMRF2 region of the EBV genome.
  • Another set of oligonucleotides (SEQ ID NOs:4-6) was designed to detect the EBNA1 region of the EBV genome.
  • oligonucleotides can be employed in individual assays for detection of the BMRF2 region of EBV (using SEQ ID NOs:1-3) and for detection of the EBNA1 region of EBV (using SEQ ID NOs:4-6).
  • the oligonucleotides can be used in a dual target assay wherein the oligonucleotides (SEQ ID NOs:1-6) simultaneously detect the BMRF2 region and the EBNA1 region of EBV within the same sample.
  • the EBV nucleic acid test was designed with two targets in mind.
  • the two targets chosen were BMRF2 and EBNA1, and are shown in FIG. 1 . These candidates could be used individually or together duplexed in a dual target assay. If used as a dual target assay, two sets of primers and probe are employed (each set detecting either EBNA1 or BMRF2). As shown in Table 1, above, the primers for BMRF2 target have the nucleic acid sequence SEQ ID NOs:1 and 3, and the probe for BMRF2 target has the nucleic acid sequence of SEQ ID NO:2.
  • the primers for EBNA1 target have the nucleic acid sequence SEQ ID NOs:4 and 6, and the probe for EBNA1 target has the nucleic acid sequence of SEQ ID NO:5.
  • the amplicon generated by the primers targeting BMRF2 is a 96 base pair long amplicon, and is shown in FIG. 2 (along with locations where the primers and probes overlap the amplicon).
  • the amplicon generated by the primers targeting EBNA1 is a 98 base pair long amplicon, and is shown in FIG. 3 (along with locations where the primers and probes overlap the amplicon).
  • EBV nucleic acid test was tested using primers/probes for detecting BMRF2 (SEQ ID NOs:1-3) and EBNA1 (SEQ ID NOs:4-6). A full process of the EBV assay was run.
  • EBV-containing samples were employed: (1) EBV-infected Raji cells extracts spiked into EBV-negative plasma; (2) extracted EBV DNA (extracted from a B95-8 cell line from Advanced Biotechnologies (Catalog No. 17-926-500)); (3) Qnostics EBV Analytic Panel (EBV1604009C); and (4) the 1 st WHO International Standard for EBV.
  • Reagents used include cobas® 6800/8800 generic PCR Master Mix, with the profile and conditions for use with the cobas® 6800/8800, and using TaqMan® amplification and detection technology.
  • the final concentration of oligonucleotides in the master mix was 0.3 ⁇ M for primers and 0.1 ⁇ M for probes.
  • the cobas® 6800/8800 PCR Profile employed is depicted in Table 2, below:
  • the 1 st WHO International Standard for EBV stock was at a concentration of 5 ⁇ 10 6 IU/ml, while the Qnostics EBV Analytic Panel has a series of samples of varying concentrations. Serial dilutions of the WHO material and an additional dilution of an aliquot of the Qnostics panel were made.
  • the Qnostics EBV Analytic Panel was analyzed at 8,000, 800, 80, and 8 IU/ml, whereas the 1 st WHO International Standard for EBV was analyzed at 800,000, 80,000, 8,000, 800, 80, and 8 IU/ml.
  • the results for the Qnostics EBV Analytic Panel and the 1 st WHO International Standard for EBV are shown in FIGS. 4 A -4C.
  • FIGS. 5 H- 5 L Another full process test of the dual target assay was done on both a cell culture derived viral material (Exact diagnostics supernatant), as depicted in FIGS. 5 H- 5 L , and a control plasmid material containing both target regions, as depicted in FIGS. 5 A -5G. These two materials were each diluted and the estimated titers overlapped at 1E4 IU/mL and 1E3 IU/mL where both materials could be tested. The resulting growth curves were shown in FIG. 5 A- 5 L . These data and results demonstrate that the primers and probes (SEQ ID NOs:1-6) amplify and detect the presence of EBV in varied sample types including commercial and international standards.
  • FIGS. 6 A and 6 B demonstrate that the positive control templates for both BMRF2 and EBNA1 are amplified and detected by the respective EBV primers/probes for BMRF2 (SEQ ID NOs:1-3) and for EBNA1 (SEQ ID NOs:4-6), but each single-target control plasmid does not cross-react with the other target, while a dual-target control plasmid and viral genomic DNA extract are amplified by both oligo sets.
  • EBV genotype 1 and EBV genotype 2 materials were used for genotype 1 and EBV genotype 2 materials.
  • genotype 1 both a cell culture derived viral material (Exact diagnostics) and a control plasmid material containing both target regions were used, the estimated titers of which overlapped.
  • genotype 2 a cell culture derived viral material (ATCC strain P3) was tested. Results are shown in FIGS. 7 and 8 . These data and results show that the assay detects and could be used for quantitation of both EBV genotype 1 and genotype 2.

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